CN117771203A - Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof - Google Patents

Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof Download PDF

Info

Publication number
CN117771203A
CN117771203A CN202311840444.1A CN202311840444A CN117771203A CN 117771203 A CN117771203 A CN 117771203A CN 202311840444 A CN202311840444 A CN 202311840444A CN 117771203 A CN117771203 A CN 117771203A
Authority
CN
China
Prior art keywords
tumor
metastasis
cells
lung
icaritin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311840444.1A
Other languages
Chinese (zh)
Inventor
刘玉萍
陈彦
孙大伟
车晓瑜
王虹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital
Original Assignee
Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital filed Critical Jiangsu Institute Of Traditional Chinese Medicine Jiangsu Integrated Traditional Chinese And Western Medicine Hospital
Priority to CN202311840444.1A priority Critical patent/CN117771203A/en
Publication of CN117771203A publication Critical patent/CN117771203A/en
Pending legal-status Critical Current

Links

Landscapes

  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses an anti-tumor and anti-tumor metastasis medicament, a preparation method and application thereof, wherein the medicament comprises extracellular microparticles, tanshinone IIA and icaritin loaded on the extracellular microparticles, and the extracellular microparticles are extracellular vesicles derived from lung metastasis tumor cells. The combination of the medicament of the invention with immunotherapy can provide an anti-tumor metastasis effect. The medicine of the invention adopts a microbubble form derived from bionic carrier cells to realize in-vivo medicine delivery, and is targeted to tumor metastasis tissues actively through metastasis targeting while simultaneously carrying tanshinone IIA and icaritin together. The cell-derived microvesicles have the advantages of high yield, large space and capability of realizing drug co-loading. The combination of the biological-derived dual-drug-carrying microbubbles and immunotherapy can significantly improve the anti-tumor metastasis effect of the immunotherapy.

Description

Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-tumor and anti-tumor metastasis medicine, and a preparation method and application thereof.
Background
Tumor metastasis is a major cause of clinical death, with about 90% of malignant tumor patients dying from distant metastasis. Tumor cells infiltrate the surrounding tissue and spread to other tissues and organs of the body through the blood system and the lymphatic system to form metastasis, ultimately leading to death of the patient. The lung is the most important metastasis target organ for all malignant tumors, and about 30% -54% of malignant tumors undergo lung metastasis in the course of natural disease. Almost 1/3 of patients dying from cancer are associated with lung metastasis. The existing means for treating tumor metastasis still mainly comprise chemotherapy, operation and radiotherapy for primary tumor, and the prior art still lacks effective treatment means for tumor metastasis due to drug resistance, adverse reaction and dose limiting toxicity.
anti-PD-1 therapies that restart the anti-tumor T cell effect hold new promise for tumor treatment, but have limited efficacy in numerous malignancies, particularly solid tumors with high invasiveness and metastatic properties, which have been shown to be closely related to the immunosuppressive microenvironment of metastatic tissue formation: (1) Abnormal blood vessels of metastatic focus tumor tissue lead to intratumoral dysplasia of Tumor Infiltrating Lymphocytes (TILs); (2) The high expression of ENPP1, the control switch for immunosuppression and metastasis, opens a huge tumor immunosuppression path, inhibits the function of TILs and promotes the metastasis capability of tumor cells.
Disclosure of Invention
Aiming at the problems in the prior art, the applicant finds that tanshinone IIA (TSA) which is an effective component in the red sage root can improve the function of endothelial cells, remarkably increase the density and the integrity of microvessels, promote the structural integrity of tumor vessels, regulate and control the normalization of the vessels and effectively improve the intratumoral infiltration of TILs; icaritin (ICT) as an effective component of herba Epimedii can inhibit migration ability of tumor cells, reduce lung nodule number, improve survival state of patients, lower expression of metastatic tumor cells ENPP1, reduce release of tumor tissue immunosuppressive adenosine, enhance function of cytotoxic T lymphocyte, and improve immunosuppressive microenvironment. Based on the above, the invention provides an anti-tumor and anti-tumor metastasis drug, and a preparation method and application thereof, wherein the drug adopts cell microparticles derived from tumor cells to encapsulate tanshinone II, so as to realize the synergistic improvement of the problem of poor intratumoral infiltration of TILs, recover the intratumoral infiltration of the TILs and inhibit the infiltration of tumor cells.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an anti-tumor and anti-tumor metastasis medicine comprises extracellular microparticles, tanshinone IIA and icaritin loaded on the extracellular microparticles, wherein the extracellular microparticles are extracellular vesicles derived from lung metastasis tumor cells.
As a preferred embodiment, the mass ratio of the tanshinone IIA to the icaritin is 1:4 to 4:1, more preferably 1:1.
As a preferred embodiment, the lung metastatic tumor cell is a lung metastatic tumor cell of melanoma, lung cancer or breast cancer.
As a preferred embodiment, the lung metastatic tumor cells are B16BL6 cells.
The invention also provides a preparation method of the anti-tumor and anti-tumor metastasis medicament, wherein the preparation method is selected from any one of a first method, a second method and a third method:
the method comprises the following steps: irradiating the lung metastasis tumor cells with ultraviolet light; adding tanshinone IIA and icaritin, incubating, and centrifuging to obtain the medicine;
the second method is as follows: irradiating lung metastasis tumor cells with ultraviolet rays, incubating, centrifuging to obtain extracellular vesicles released by the lung metastasis tumor cells, and incubating the extracellular vesicles with tanshinone IIA and icaritin;
and a third method: irradiating lung metastasis tumor cells with ultraviolet rays, incubating, centrifuging to obtain extracellular vesicles released by the lung metastasis tumor cells, mixing the extracellular vesicles with tanshinone IIA and icaritin, performing ultrasonic treatment, and standing for recovery.
Preferably, the ultraviolet irradiation time is 1h.
Preferably, the incubation time is 12 hours.
Preferably, the co-incubation time is 2h.
Preferably, the centrifugation method is as follows: and centrifuging under the centrifugal force of 300-14000 g.
Preferably, the second method further comprises the step of removing the supernatant after co-incubation and the non-entrapped drug.
Preferably, the conditions of the ultrasonic treatment are as follows: the ultrasonic treatment is carried out for 4 seconds with the amplitude of 20%, the intermittent treatment is carried out for 2 seconds, and the total circulation is 6 times with 2 minutes as one circulation.
Preferably, the conditions for the rest recovery are: recovery was carried out at 37℃for 2h.
The invention further provides application of the anti-tumor and anti-tumor metastasis medicament in preparing PD-1 inhibition treatment auxiliary agent.
The invention combines tanshinone IIA and icaritin according to a specific proportion and prepares the drug-containing microparticles, which can be delivered to a focus area of a metastasis, and can obviously improve the curative effect of the PD-1 inhibitor on tumor metastasis when being combined with the PD-1 inhibitor.
The structural formula of tanshinone IIA (TSA) is as follows:
icaritin (ICT) has the structural formula:
the invention has the beneficial effects that:
the micro-particles from tumor cells are used for encapsulating tanshinone IIA and icaritin, so that the intratumoral infiltration effect of TILs is recovered, and the immunosuppression tumor microenvironment is improved. Wherein tanshinone IIA regulates and controls vascular normalization by improving endothelial cell function, and improves intratumoral infiltration of TILs. Icaritin reduces the release of tumor tissue immunosuppressive adenosine, enhances the function of cytotoxic T lymphocytes and improves the immunosuppressive microenvironment by down regulating ENPP1 expression of tumor cells. The two medicines are combined with the PD-1 inhibitor to obviously improve the curative effect of the PD-1 inhibitor on tumor metastasis.
Drawings
FIG. 1 is an inverted micrograph showing the horizontal migration of B16BL6 cells under the influence of the different icaritin dose groups and the control group in example 1.
Fig. 2 is the amount of B16BL6 cell level migration under the influence of the different icaritin dose groups and the control group in example 1, (n=3,)；
fig. 3 shows the inhibition ratios of CTLL-2 cells under co-culture conditions by icaritin groups and control groups at different concentrations in example 2, (n=6,). And (3) injection: comparison with blank control group p<0.01。
FIG. 4 is an inverted micrograph of cells under the influence of tanshinone IIA at various concentrations and blanks in example 3.
Fig. 5 shows the relative cellular activities of the vascular endothelial cells of bond.3 mice at various concentrations of tanshinone IIA and the blank influence in example 3 (n=6,)。
FIG. 6 is a photograph of fluorescent inverted microscope of lower chamber liquid obtained under the influence of tanshinone IIA at different concentrations and blank in example 4.
Fig. 7 shows FITC fluorescence intensity of lower chamber liquid in each set of samples measured in example 4, (n=3,)
FIG. 8 is a graph showing the particle size distribution of TSA & ICT-TMPs obtained in example 5.
FIG. 9 is a scanning electron microscope image of TSA & ICT-TMPs and extracellular microparticles obtained in example 5.
FIG. 10 shows the TSA in example 6 in the TSA-ICT group&Migration of CTLL-2T lymphocytes across bond.3 mouse endothelial cells under the influence of ICT-TMPs, TSA, ICT and blank (control) (n=6,). And (3) injection: comparison with TSA-ICT p<0.05。
FIG. 11 shows the results of TSA-ICT and TSA in example 7&Inhibition of CTLL-2 cells under co-culture conditions under the influence of ICT-TMPs, extracellular Microparticles (MPs) and control (n=6,) The method comprises the steps of carrying out a first treatment on the surface of the And (3) injection: comparison with control p<0.001，*p<0.05。
Fig. 12 is a graph showing the change in body weight of mice with lung metastasis of B16BL6 melanoma treated with each of the drug groups and the blank group in example 8.
Figure 13 is a graph of the number of node points in mice following treatment of B16BL6 melanoma lung metastasis mice with each group of drug and blank in example 8 (n=6,). And (3) injection: comparison with model group p<0.001，**p<0.01，*p<0.05。
Figure 14 is an abnormal tumor weight of mice after treatment of B16BL6 melanoma lung metastasis mice with each group of drug and blank in example 8 (n=6,)。
fig. 15 is a representative picture of the lungs of mice following pulmonary metastasis of mice in example 8 using each group of drugs and blank treatment B16BL6 melanoma.
FIG. 16 is a graph of mouse lung tissue CD4 after lung metastasis with each group of drugs and blank treatment B16BL6 melanoma in example 8 + T-cell immunofluorescence staining results (Scale bar:100 μm).
FIG. 17 is a graph of CD8 lung tissue of mice after lung metastasis with each group of drugs and blank treatment B16BL6 melanoma in example 8 + T-cell immunofluorescence staining results (Scale bar:100 μm).
FIG. 18 shows the results of immunohistochemical staining of mouse lung tissue ENPP1 (Scale bar:100 μm) following lung metastasis using the various groups of drug and blank treatment B16BL6 melanoma in example 8.
Detailed Description
Examples relate to drugs, reagents, cells, instrument sources as follows:
experimental cells: CTLL-2 mouse T cells, jurkat human T lymphoblast cells, H 22 -Luc mouse liver cancer cells, HUVEC human umbilical vein endothelial cells, bend.3 mouse brain microvascular endothelial cells, all purchased from Shanghai cell bank, national academy of sciences.
Experimental animals: SPF grade, male C57BL/6 mice, 60, 4 weeks old, purchased from Jiangsu Jiuyaokang Biotechnology Co., ltd. Experimental animal license number: SCXK (Su) 2023-0009. The experimental animals were kept in a barrier environment of the experimental animal center of the institute of traditional Chinese medicine, jiangsu province [ license number for use by experimental unit: SYXK 2016-0018, the experiment strictly complies with the ethical relevant regulations of the experimental animal. The illumination time is 12 hours, the temperature is constant (20-26 ℃), the humidity is constant (40% -70% RH), the water is freely drunk, and the complete standard feed is taken. Experiments were started 7 days after the adaptive feeding.
Medicine and reagent: tanshinone IIA and astragaloside IV (HPLC is more than or equal to 98 percent), and the source organisms are selected from the group consisting of the following raw materials; tween 80, tetramethylazoblue (MTT), collagenase i, collagenase iv, dnase i, hyaluronidase (Biofroxx); zombieAqua Fixable Viability kit, APC-cy7 anti-mouse CD45, APC anti-mouse CD3, PE-cy7 anti-mouse CD4, PE anti-mouse CD8 (Biolegend); murine PD-1 antibody for injection (Bioxcell); glycerol tripropionate, feCl 3 ·6H 2 O、Fe(NO 3 ) 3 ·9H 2 O (ala Ding Shiji); anhydrous sodium acetate, nitric acid, hydrofluoric acid (south Beijing chemical reagent); dimethyl sulfoxide DMSO, diethylene glycol (Sigma); sodium acrylate, trimesic acid, terephthalic acid, 2-amino terephthalic acid, potassium fluorescein salt, cholesterol (rohn reagent); protease inhibitors (csnphara); RPMI1640 medium (basal media); DMEM medium (Gibco); fetal bovine serum (Deary Tech); CCK8 (syn chemical); mitomycin C (GLPBIO Co.); matrigel, 6-well 0.4 μmTorrandwell cell, 24-well 5 μmTorrandwell cell, 24-well 8 μmTorrandwell cell (Corning), polyoxyethylene 40 hydrogenated castor oil (RH 40), polyethylene glycol 15 hydroxystearate (HS 15) (Pasteur (China) Inc.), polyethylene glycol-300 (PEG-300), polyethylene glycol-400 (PEG-400), ethyl oleate, isopropyl myristate, triacetin, tributyrin (national drug group). Other chemical reagents are analytically pure unless otherwise specified.
Instrument: mutisman Go Go type enzyme labelling instrument, ST16R type centrifuge, 311 type CO 2 Incubator (thermo fisher); multifunctional enzyme labelling instrument (PerkinElmer company); flow cytometry (CytoFLEX BE42432, beckman, usa); ultra clean bench (Su Antai air technologies limited); a cytometer (IC 1000 type, countstar); MS205DU type electronic balance in ten thousandths (Mettler Toledo); XMTD-8222 high temperature oven (fine macro instrument); OS20-Pro type mechanical stirrer (SCILogex); ABM type small animal gas inhalation anesthesia machine (jade grinding instrument); chemStudio PLUS type small animal in vivo imager (analytical JenaAG).
Example 1: effect of icaritin at different concentrations on B16BL6 tumor cell level migration
When the fusion degree of the B16BL6 cells is 80-90%, the single cells are digested by trypsin and resuspended to 3X 10 per well 5 Individual cells were seeded in 6-well plates at 37℃with 5% CO 2 Culturing in the environment until the cells adhere to the wall to form a monolayer of cells. The 10. Mu.L gun head was scored vertically on the cell culture plate along the 6-well plate diameter, and after 3 gentle flushes of PBS, the recordings were taken (0 h). Experimental group a blank control group (blankk) The samples of each group were added to the cell culture solution and cultured for 24 hours, and then photographed under an inverted microscope, and photographs of 0 hours and 24 hours are shown in FIG. 1, as solvent control group (dmso), icaritin dose group (5 to 40 μm). And calculating the change of the healing area of the scratch, and analyzing the migration condition of the cell.
The results show that: the icaritin has obvious inhibition effect on the level migration of B16BL6 cells (as shown in figure 2) at the dosage of 5-40 mu m, which indicates that the icaritin has a certain inhibition effect on the transfer capacity of tumor cells.
Example 2: effect of icaritin at different concentrations on CTLL-2 cell Activity under Co-culture conditions
The upper chamber was seeded with B16BL6 cells (5X 10) 4 Number/well), the lower chamber was seeded with CTLL-2 mouse T cells (1X 10) 5 Per well), CTLL-2 and B16BL6 cell co-culture models were established. The tumor cells in the upper chamber are treated for 24 hours by using a blank control group (control) and icaritin groups with different concentrations (0.2-4 mug/mL). After 24h, the ventricular CTLL-2 cells were removed for counting and activity detection.
The results are shown in FIG. 3: icaritin has a certain inhibition effect on T cells, and the icaritin with the concentration of 0.4 mug/mL has the best inhibition effect, and the influence on the T cells gradually decreases with the increase of the concentration.
Example 3: effect of tanshinone IIA at different concentrations on vascular endothelial cell proliferation in bond.3 mice
The bond.3 cells in logarithmic growth phase were taken at 1X 10 4 The cells were inoculated in 96-well plates with 6 parallel wells each, and the cells were incubated in DMEM medium containing 10% fetal calf serum, 5% CO 2 After culturing in a cell incubator at 37 ℃ for 24 hours, tanshinone IIA with different concentrations and blank (blank) are added, culturing is continued for 24 hours, and photographing is carried out under an inverted microscope, so that the chart shown in figure 4 is obtained. After incubation, 10 μl of CCK8 reagent was added to each well, incubation was continued for 1.5h, absorbance was measured at a wavelength of 450nm on a microplate reader, and cell activity was calculated from the absorbance.
Relative cell activity= (dosing well absorbance-medium absorbance)/(blank absorbance-medium absorbance).
The results are shown in FIG. 5: tanshinone IIA has a certain inhibition effect on bond.3 cells, the tanshinone IIA with the concentration of 0.25 mug/mL and 0.5 mug/mL has the best inhibition effect, and the effect on proliferation of mouse endothelial cells is smaller along with the increase of the concentration.
Example 4: effect of varying concentrations of tanshinone IIA (TSA) on endothelial cell leakage
The Transwell upper chamber is inoculated with 5×10 5 HUVEC cells were grown in individual cells/well to form a dense monolayer, blank medium was used as Blank for the lower chamber, and hepatoma cell culture supernatant was added to the lower chamber of the experimental group. Then, the upper chambers of each experimental group and the Blank group were replaced with DMEM complete culture solution containing tanshinone iia at different concentrations (TSA was not added to the upper chamber of the Blank group), and after 24 hours, the upper chamber was replaced with FITC-Dextran solution at 0.1mg/mL, and after 1 hour, the lower chamber liquid was collected, photographed with a fluorescence inversion microscope, and the fluorescence intensity was detected to evaluate the effect of the drug on vascular endothelial cell leakage.
The results are shown in FIGS. 6-7: when the concentration of tanshinone IIA is 2 mug/mL, compared with an experimental group without TSA, the fluorescence intensity of FITC of lower chamber liquid is the lowest, which shows that tanshinone IIA has obvious improvement effect on endothelial cell leakage caused by tumor microenvironment.
Example 5: the invention relates to preparation and characterization of anti-tumor and anti-tumor metastasis medicaments
The influence of different dosing prescriptions and different preparation methods on drug-containing microparticles (TSA & ICT-TMPs) carrying tanshinone IIA and icaritin together is tested in the embodiment.
(1) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 1:1, incubating in incubator for 12 hr, collecting cell culture supernatant, centrifuging at 300g and 4deg.C for 5min, at 1300rpm and 4deg.C for 8min, centrifuging at 14000g and 4deg.C for 2min, discarding precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, collecting precipitate, suspending in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain the final productTo a microparticle precipitation containing TSA and ICT, i.e. TSA&ICT-TMPs。
(2) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 1:2 in the final product, continuously incubating for 12 hr in an incubator, collecting cell culture supernatant, centrifuging at 1300rpm and 4deg.C for 5min, centrifuging at 14000g and 4deg.C for 8min, centrifuging at 14000g and 4deg.C for 2min, discarding the precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, collecting precipitate, re-suspending in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain TSA-containing micro-particle precipitate&ICT-TMPs。
(3) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 1:4 in the final product, continuously incubating for 12 hr in an incubator, collecting cell culture supernatant, centrifuging at 1300rpm and 4deg.C for 5min, centrifuging at 14000g and 4deg.C for 8min, centrifuging at 14000g and 4deg.C for 2min, discarding the precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, collecting precipitate, re-suspending in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain TSA-containing micro-particle precipitate&ICT-TMPs。
(4) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 2:1, incubating in incubator for 12 hr, collecting cell culture supernatant, centrifuging at 1300rpm and 4deg.C for 5min, centrifuging at 14000g and 4deg.C for 2min, discarding precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, suspending the precipitate in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain TSA-containing microparticle precipitate&ICT-TMPs。
(5) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 4:1, incubating in incubator for 12 hr, collecting cell culture supernatant, centrifuging at 1300rpm and 4deg.C for 5min, centrifuging at 14000g and 4deg.C for 2min, discarding precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, suspending in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain TSA-containing microparticle precipitate&ICT-TMPs。
(6) Method one preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 Irradiating with ultraviolet for 1 hr, adding tanshinone IIA and icaritin according to the mass ratio of 1.25:1, continuously incubating in an incubator for 12 hr, collecting cell culture supernatant, centrifuging at 300g and 4deg.C for 5min, at 1300rpm and 4deg.C for 8min, centrifuging at 14000g and 4deg.C for 2min, discarding the precipitate, collecting supernatant, centrifuging at 14000g and 4deg.C for 60min, collecting precipitate, re-suspending in PBS, centrifuging at 14000g and 4deg.C for 60min to obtain micro-particle precipitate containing TSA and ICT, i.e. TSA&ICT-TMPs。
(7) Preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 And (3) continuously incubating for 12 hours in an incubator for 1 hour by ultraviolet irradiation, collecting cell culture supernatant, centrifuging for 5 minutes at 300g and 4 ℃, centrifuging for 8 minutes at 1300rpm and 4 ℃, centrifuging for 2 minutes at 14000g and 4 ℃, discarding the precipitate, collecting supernatant, centrifuging for 60 minutes at 14000g and 4 ℃, collecting the precipitate to be resuspended in a proper amount of PBS, and centrifuging for 60 minutes at 14000g and 4 ℃ to obtain blank microparticle precipitates, namely TMPs. Incubating blank microparticles with tanshinone IIA and icaritin for 2h at 37deg.C according to the mass ratio of tanshinone IIA to icaritin of 1:1, centrifuging at 14000g, and removing supernatant and non-entrapped medicine to obtain TSA and ICMicroparticle precipitation of T, i.e. TSA&ICT-TMPs。
(8) Method three preparation of TSA&ICT-TMPs: about 4X 10 of B16BL6 cells in logarithmic growth phase 7 And (3) continuously incubating for 12 hours in an incubator for 1 hour by ultraviolet irradiation, collecting cell culture supernatant, centrifuging for 5 minutes at 300g and 4 ℃, centrifuging for 8 minutes at 1300rpm and 4 ℃, centrifuging for 2 minutes at 14000g and 4 ℃, discarding the precipitate, collecting supernatant, centrifuging for 60 minutes at 14000g and 4 ℃, collecting the precipitate to be resuspended in a proper amount of PBS, and centrifuging for 60 minutes at 14000g and 4 ℃ to obtain blank microparticle precipitates, namely TMPs. And correspondingly carrying out ultrasonic treatment on blank microparticles, tanshinone IIA and icaritin in an ice water bath according to the mass ratio of tanshinone IIA to icaritin in a final product of 1:1, wherein the ultrasonic treatment is carried out under the condition that: 20% amplitude, 4s on, 2s off, 2min circulation, total circulation for 6 times, taking out, placing in a 37 deg.C incubator, recovering for 2 hr, centrifuging at 14000g, removing supernatant and non-entrapped medicine to obtain micro-granule precipitate containing TSA and ICT, namely TSA&ICT-TMPs。
FIG. 8 shows the particle size distribution of the TSA & ICT-TMPs produced.
Example 6: effect of TSA & ICT-TMPs on CTLL-2T lymphocyte behavior across Bend.3 mouse endothelial cells
Using Transwell nesting, containing 24 well plates (PC membrane, 6.5mm, pore size 5.0 μm), the bond.3 cells were first resuspended in medium containing 10% fbs RPMI1640 at a cell density of 1×10 5 inoculating/mL onto a Transwell cell membrane, culturing in a lower chamber with 500 μl of RPMI1640 medium containing 10% FBS, and adding 5% CO 2 Culturing in a 37℃cell incubator, and adding the TSA prepared in example 5 (6) to the upper chamber&ICT-TMPs (additive amount is calculated by TSA0.18 mu g/mL), TSA-ICT (ICT 0.16 mu g/mL, TSA0.18 mu g/mL), TSA (0.18 mu g/mL) and ICT (0.16 mu g/mL), and the cells are cultured for 24 hours for fusion of 80% for later use by taking the group without adding the medicament as Control. CTLL-2 cell suspension was prepared using 2% FBS RPMI1640 medium, and the cell density of each group was adjusted to 5X 10 6 mu.L of the above cell suspension was added to the upper chamber, 500. Mu.L of RPMI1640 medium containing 20% FBS was added to the lower chamber, and 5% CO was added 2 、37℃The cells were cultured in a cell incubator for 3h to allow the cells to move along the porous membrane gaps from top to bottom. After 3 hours, the cells were removed, the liquid in the lower chamber was sucked to gently blow the lower layer of the porous membrane, then the medium in the lower chamber was sucked out entirely, centrifuged at 1000rpm for 5 minutes, resuspended in 100. Mu.L medium, counted 3 times and averaged. Based on the counting result, the number of the migration cells was calculated.
As shown in FIG. 10, TSA & ICT-TMPs can promote transendothelial migration of T lymphocytes.
Example 7: effect of TSA & ICT-TMPs on CTLL-2 cell Activity under Co-culture conditions
The upper chamber was seeded with B16BL6 cells (5X 10) 4 Number/well), the lower chamber was seeded with CTLL-2 mouse T cells (1X 10) 5 Per well), CTLL-2 and B16BL6 cell co-culture models were established. TSA prepared in example 5 (6), respectively&ICT-TMPs (additive amount was calculated as TSA 0.18. Mu.g/mL), extracellular microparticles (MPs, 1000. Mu.g/mL), TSA-ICT (ICT 0.16. Mu.g/mL, TSA 0.18. Mu.g/mL), and drug-free blank (Control) were used to treat upper chamber tumor cells for 24h. After 24h, the ventricular CTLL-2 cells were removed for counting and activity detection.
As shown in FIG. 11, the inhibition rate of CTLL-2 cells was the lowest under the influence of TSA-ICT-MPs, i.e., the activity of mouse T lymphocytes under co-culture conditions was improved by TSA-ICT-MPs.
Example 8: pharmacodynamic experiments for treating B16BL6 melanoma lung metastasis using TSA-ICT-MPs in combination with PD-1 inhibitors
Culturing B16F10-Luc melanoma cells under cell conditions, centrifuging the cells when they are in logarithmic phase, adding PBS, and re-suspending to give 5×10 per ml 5 Each C57BL/6 male mouse tail was inoculated intravenously with 0.2mL of the cell suspension for molding, and after about 14 days melanoma developed pulmonary metastasis. Experiment TSA prepared in example 5 (6)&ICT-TMPs。
Mice were randomized into 7 groups by body weight, into blank (saline only), model (saline only), microparticles (extracellular microparticles only), TSA-ICT (TSA-ICT only), TSA-ICT-microparticles (TSA & ICT-TMPs only), alpha-PD-1 (alpha-PD-1 only), combination (TSA & ICT-TMPs and alpha-PD-1). Each group was given by tail intravenous injection of 0.2mL of physiological saline, 0.2mL of MPs (1000. Mu.g/mL), 0.2mL of the drug (additive amount was calculated as TSA 0.18. Mu.g/mL), once every two days, the group alpha-PD-1 was given by intraperitoneal injection at a dose of 5mg/kg of PD-1 inhibitor, the group TSA & ICT-microparticles+alpha-PD-1 was given by intraperitoneal injection at a dose of 5mg/kg of PD-1 inhibitor, and at the same time by tail intravenous injection at a dose of 0.2mL of the drug (additive amount was calculated as TSA 0.18. Mu.g/mL), once every two days and the body weights of the mice were recorded. 14 days after administration, the eyeballs were taken for blood collection, the mice were sacrificed by neck removal, lung melanoma was dissected, and lung node number, abnormal lung weight and lung tumor fluorescence were measured and analyzed, and tumor tissues were subjected to flow cytometry analysis and histological analysis.
The results are shown in FIGS. 12-15:
TSA & ICT-MPs used in combination with PD-1 inhibitors showed significantly lower lung node numbers, abnormal tumor weights, and lung tumor fluorescence intensities than model (P < 0.05) and TSA & ICT-microparticles+α -PD-1 lung node numbers, abnormal tumor weights, etc. than α -PD-1 and TSA & ICT-microparticles+α -PD-1 mice had greater body weights than model and other groups (FIGS. 12-14).
Lung tissue from TSA & ICT-microparticle + a-PD-1 group showed significantly reduced lung metastases compared to model group and a-PD-1 group.
(FIG. 15)
Immunofluorescence results showed that TSA compared to model group&CD4 in ICT-microparticle+alpha-PD-1 group mouse lung tissue tumor + Infiltration was increased (fig. 16).
Immunofluorescence results showed that TSA-ICT-MPs combined with PD-1 inhibitors used in mice lung tissue tumors showed CD8 compared to model group + Infiltration was increased (fig. 17). The immunohistochemical results showed that TSA compared to model group&ICT-microparticles+α -PD-1 group mice had reduced expression of lung tumor ENPP1 (FIG. 18).
The result shows that the tanshinone IIA-icaritin drug-containing microparticle system can inhibit the lung metastasis of melanoma, and the drug-containing microparticle system is cooperated with the PD-1 inhibitor, so that the anti-tumor metastasis curative effect of the PD-1 inhibitor is effectively improved. In addition, the drug-containing microparticles cooperate with the PD-1 inhibitor to effectively improve the level of lymphocytes in lung metastasis tumor, increase the infiltration of anti-tumor T lymphocytes, enhance the activity of the infiltrated T lymphocytes, improve the microenvironment of tumor immunosuppression and inhibit tumor metastasis.

Claims (10)

1. An anti-tumor and anti-tumor metastasis drug is characterized by comprising extracellular microparticles, tanshinone IIA and icaritin loaded on the extracellular microparticles, wherein the extracellular microparticles are extracellular vesicles derived from lung metastasis tumor cells.
2. The medicament according to claim 1, wherein the mass ratio of tanshinone IIA to icaritin is 1:4-4:1, preferably 1:1.
3. The medicament of claim 1, wherein the lung metastatic tumor cells are lung metastatic tumor cells of melanoma, lung cancer or breast cancer.
4. A medicament according to claim 1 or 3, characterized in that the lung metastatic tumor cells are B16BL6 cells.
5. The method for preparing the anti-tumor and anti-tumor metastasis medicament according to any one of claims 1-4, wherein the preparation method is selected from any one of a first method, a second method and a third method:
the method comprises the following steps: irradiating the lung metastasis tumor cells with ultraviolet light; adding tanshinone IIA and icaritin, incubating, and centrifuging to obtain the medicine;
the second method is as follows: irradiating lung metastasis tumor cells with ultraviolet rays, incubating, centrifuging to obtain extracellular vesicles released by the lung metastasis tumor cells, and incubating the extracellular vesicles with tanshinone IIA and icaritin;
and a third method: irradiating lung metastasis tumor cells with ultraviolet rays, incubating, centrifuging to obtain extracellular vesicles released by the lung metastasis tumor cells, mixing the extracellular vesicles with tanshinone IIA and icaritin, performing ultrasonic treatment, and standing for recovery.
6. The method according to claim 5, wherein the ultraviolet irradiation time is 1h.
7. The method of claim 5, wherein the incubation time is 12 hours; the incubation time was 2h.
8. The method of claim 5, wherein the centrifugation is performed by: and (3) centrifuging under the centrifugal force condition of 300-14000 g.
9. The method according to claim 5, wherein the ultrasonic treatment conditions are: the ultrasonic treatment is carried out for 4 seconds with the amplitude of 20%, the intermittent treatment is carried out for 2 seconds, and the total circulation is 6 times with 2 minutes as one circulation.
10. Use of an anti-tumor and anti-tumor metastasis medicament according to any one of claims 1-4 in the preparation of a PD-1 inhibitory therapeutic adjuvant.
CN202311840444.1A 2023-12-29 2023-12-29 Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof Pending CN117771203A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311840444.1A CN117771203A (en) 2023-12-29 2023-12-29 Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311840444.1A CN117771203A (en) 2023-12-29 2023-12-29 Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN117771203A true CN117771203A (en) 2024-03-29

Family

ID=90394116

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311840444.1A Pending CN117771203A (en) 2023-12-29 2023-12-29 Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN117771203A (en)

Similar Documents

Publication Publication Date Title
EP2794859B1 (en) Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
US9844508B2 (en) Tumor vaccine and method for producing the same
US10548853B2 (en) Oncolytic virus formulation and preparation method thereof
EP1930414B1 (en) Method for activation treatment of antigen-presenting cell
CN106727331B (en) Composition of immunoliposome-cycloddinucleotide, preparation method and application of immunoliposome-cycloducleotide in resisting tumor
Dai et al. Nanomedicines modulating myeloid-derived suppressor cells for improving cancer immunotherapy
Wen et al. Nanovaccines fostering tertiary lymphoid structure to attack mimicry nasopharyngeal carcinoma
CN106222141A (en) NK cell culture fluid and cell culture processes
CN110029088B (en) Tumor cell apoptosis corpuscle and preparation method and application thereof
Zhao et al. Combining photothermal ablation-based vaccine with immune checkpoint blockade for synergistic osteosarcoma immunotherapy
US8691568B2 (en) Method for preparing cell populations with anti-tumor immune response activity
CN114191539B (en) Exosome nano particle for compositely co-carrying small molecule nucleic acid and active protein, and preparation method and application thereof
CN117771203A (en) Anti-tumor and anti-tumor metastasis medicine, and preparation method and application thereof
CN115612670A (en) Method for preparing tumor cell microparticles by microwave
CN114748424B (en) Liposome drug delivery system and preparation method and application thereof
FR2615737A1 (en) PREPARATION AND USE OF INTERFERON-GAMMA
Yang et al. Engineered antler stem cells derived exosomes potentiate anti-tumor efficacy of immune checkpoint inhibitor by reprogramming immunosuppressive tumor microenvironment
CN114349950A (en) Antibody synthetic bacterium-nano stimulant hybrid system and application thereof
Hu et al. Anti-tumor effects of fusion vaccine prepared by renal cell carcinoma 786-O cell line and peripheral blood dendritic cells of healthy volunteers in vitro and in human immune reconstituted SCID mice
JP2023537161A (en) Method for producing universal immune cells and application thereof
CN106119192B (en) Composition and its application in CIK cell culture
CN116617222B (en) Application of small molecular ion channel blocker MK-801 in preparation of medicines for treating tumors or resisting infection
CN117402218B (en) Individualized dendritic cell vaccine for Survivin positive tumor and preparation method thereof
CN113648404B (en) Tumor whole-cell targeting reagent and preparation method and application thereof
CN118001287A (en) Antitumor traditional Chinese medicine composition and preparation thereof and application of antitumor traditional Chinese medicine composition in preparation of PD-1 inhibitor synergist

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination